The invention is in the field of electrophoresis. It is of particular interest in terms of its applications in genetic engineering and molecular biology.
The invention which is based upon the discovery of a new kind of electrophoresis makes it possible, inter alia, to carry out important analyses which were not possible or practical with previously known techniques. Potential applications include the separation of chromosomal DNA, chromosomal mapping, the convenient production of genetic libraries, studies on the effects of various drugs on chromosomal DNA, and the convenient characterization of polymers. The invention makes it possible to separate with a high degree of resolution and at high speeds larger particles (molecules) than those capable of resolution with prior art techniques, to concurrently separate particles which differ substantially in mass. In a preferred embodiment the invention makes it possible to lyse cells for electrophoretic separation of macromolecules contained in the cells with minimal degradation or breakage.
Electrophoresis in which particles such as a mixture of macromolecules are moved, e.g., through a gel matrix, by an electric field, is a widely used technique for qualitative analysis and for separation, recovery and purification. It is particularly important in the study of proteins, nucleic acids and chromosomes. See, e.g., Cantor, C. R. et al., Biophysical Chemistry, Freeman, 1980, Part 2, pp. 676, 683. Indeed, it is probably the principal tool used in most DNA and chromosomal analysis.
Difficulties arise when electrophoretic separation of very large particles is attempted. For example, using previously known techniques, the size of the largest DNA molecule routinely handled is that of a bacteriophage (3.2.times.10.sup.7 daltons). Such a limit on size prevents many kinds of desirable analyses from being carried out. For example, intact chromosomal DNAs are larger and are typically reduced in size in order to make it possible to work with them. This, however, destroys important information encoded within the DNA and precludes many important experiments.
It has been proposed to extend gel electrophoresis to particles of higher mass by reducing the gel concentrations. However, this adversely affects resolution, makes experimental conditions difficult to control and has not been successfully applied to DNA molecules having molecular weights greater than about 5.times.10.sup.8 daltons. Fangman, W. L., Nucleic Acids Research, Vol. 5, No. 3, March 1978, pp. 653-655; Serwer, P., et al., Electrophoresis, 1981, Walter, deGreuyter and Coe, pp. 237-243.
It is believed that resolution in previously known electrophoresis techniques is field-dependent since lower electric field intensities generally give higher resolution. As a consequence, electrophoresis runs in which higher resolution is desired often take as long as 100 hours. Moreover, particle mobility, and hence resolution capability, is believed to vary with the logarithm of the mass of the particles to be separated, which of course is not a highly sensitive basis for obtaining separations. Additionally, in known prior art gel electrophoresis, different gel concentrations are typically used for different mass or molecular weight ranges, thereby limiting the range of particles which can be concurrently resolved. Furthermore, previously known electrophoresis techniques are typically used to separate only small amounts of particles, and the process cannot conveniently be extended to larger amounts.
Despite the fact that electrophoresis has been used for some time, and despite the fact that important limitations thereof and the need to overcome them have also been long known, no previous proposals are known which have successfully overcome such limitations.
This invention is a significant departure from the established principles of electrophoresis and is based on the surprising discovery that electrophoresis through deliberately varied electric fields, rather than through the uniform fields sought in previously known electrophoresis methods, unexpectedly yields highly desirable results. More specifically, the invention is based on the discovery that desirable separation results when particles are subjected to respective electrical fields which move them in overall directions generally transverse to the respective general directions of the fields. Particularly desirable results are achieved in at least those cases examined to date when at least one of the electric fields has a deliberate intensity gradient in a direction transverse to its own. As a specific nonlimiting example, two fields can be used which alternate between respective high and low intensities out of phase with each other and are in directions transverse to each other. For example, one of the fields can be on while the other one is off, etc. Particularly good results are obtained when the on and off times of the fields are related to the mass of the particles to be separated, e.g., when the on and off periods are proportional to the mass of the particles raised to a power of about 1.5.
One of the important advantages of this discovery is that it dramatically extends the mass range of particles which can be electrophoretically separated at high resolution. As a nonlimiting example, the new technique can separate at high resolution particles whose mass is about 1.2.times.10.sup.9 daltons, while the upper limit of previously known methods which provide lower resolution, is believed to be about 0.5.times.10.sup.9 daltons. It is believed that the new technique can also resolve particles larger than 1.2.times.10.sup.9 daltons. Another important advantage is that in the new technique resolution is much less dependent on electric field intensity; consequently, the new kind of electrophoresis can be run at much higher speed, so long as heat produced can be effectively dissipated. As a result, a typical laboratory run can be carried out in 4 to 8 hours, while corresponding runs using prior art techniques require 12 to 100 hours. Another significant advantage of the new technique is that larger amounts of sample, as compared to the known prior art, can be used, thus giving increased resolution and sensitivity. A further advantage is that the new technique can simultaneously resolve, in the same gel, particles from a wider mass range than is believed possible with prior art techniques. As a nonlimiting example, the new technique can resolve simultaneously, in the same gel, particles ranging in mass from about 10.sup.6 to about 10.sup.9 daltons. With previously known techniques several different gel concentrations would have been required to resolve particles in the narrower mass range from about 10.sup.6 to about 10.sup.8 daltons. As yet another important aspect of the invention, a technique has been found to minimize handling damage to cell-derived macromolecules by lysing cells or spheroplasts in a block of gel which is the same as, or compatible with, the electrophoresis gel, and implanting the entire block in the electrophoresis chamber.
These and other advantages of the invention, as well as additional inventive features, will become apparent from the detailed description which follows.